Nucleic acids encoding IL13 mutants

ABSTRACT

This invention provides nucleic acid molecules encoding mutant human interleukin 13 molecules showing varying specificity for the restricted (IL4 independent) IL13 receptor. The mutant hIL13 molecules include those made by substituting the amino acid residues that occur in the alpha-helix regions of native hIL13 with various other amino acid residues. Some of the mutants retain the ability to bind and cause signaling through IL13 receptors, while other mutants do not.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/053,406 filed Jan. 17, 2002, now allowed, which isdivisional of U.S. patent application Ser. No. 09/679,710 filed on Oct.5, 2000, now U.S. Pat. No. 6,576,232, which is a continuation-in-part ofU.S. patent application Ser. No. 09/054,711 filed on Apr. 3, 1998, nowU.S. Pat. No. 6,296,843, and is related to and claims the benefit ofU.S. Provisional patent application No. 60/157,934 filed on Oct. 6,1999.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made in part with Government support under grantCA741145 awarded by the National Institutes of Health. The Governmentmay have certain rights in the invention.

BACKGROUND OF THE INVENTION

Human interleukin 13 (hIL13) is a 114 amino acid cytokine secreted byactivated T cells. Minty et al. (1993) Nature, 362:248-250; and McKenzieet al. (1993) Proc. Natl. Acad. Sci. USA, 90:3735-3739. hIL13 isinvolved in regulating several different physiological responses. Amongthese, hIL13 has been shown to downregulate the production of cytokinesinvolved in inflammation. Minty et al., supra; and de Waal Malefyt etal. (1993) J. Immunol., 151:6370-6381. It has also been shown toupregulate expression of major histo-compatibility class II moleculesand CD23 on monocytes, and to regulate various aspects of B cellfunction De Waal Malefyt et al. (1993) Res. Immunol. 144:629-633;McKenzie et al., supra; and de Waal Malefyt et al. (1993) J. Immunol.,151:6370-6381. In addition to regulating cells of the immune system,IL-13 has also been shown to act on other cell types. For example, IL13has been shown to modulate expression of vascular cell adhesionmole-cule-1 (VCAM-1) on endothelial cells. Sironi et al. (1994) Blood,84:1913-1921; Bochner et al. (1995) J. Immunol., 154:799-803; andSchnyder et al. (1996) Blood, 87:4286-4295.

Based on its predicted secondary structure, hIL13 has been added to agrowing family of growth hormone-like cytokines that all exhibit bundledaa-helical core topology. Bamborough et al. (1994) Prot. Engin.,7:1077-1082. Structural analyses indicated that hIL13 is a globularprotein comprised mainly of four aa-helical regions (helices A, B, C,and D) arranged in a “bundled core.” Miyajima et al. (1992) Ann. Rev.Immunol., 10, 295-331.

While dissimilar at the primary amino acid level, hIL13 and humaninterleukin 4 (hIL4) bind and signal through a shared receptor complex.Zurawski et al. (1993) EMBO J., 12:2663-2670; and Tony et al. (1994)Eur. J. Biochem., 225:659-66. This shared receptor is a heterodimer thatincludes a first subunit of approximately 140 kDa termed p140, and asecond subunit of approximately 52 kDa termed α′ or IL13Rα1. Idzerda etal. (1990) J. Exp. Med., 173:861-873; Obiri et al. (1995) J. Biol.Chem., 270:8797-8804; Hilton et al. (1996) Proc. Natl. Acad. Sci. USA,93:497-501; and Miloux et al. (1997) FEBS Letters, 401:163-166. UnlikehIL4, hIL13 does not bind p140 in the absence of α′. Vita et al. (1995)J. Biol. Chem., 270:3512-3517. In addition to the shared receptor,another hIL13 receptor termed the restricted (IL4 independent) receptorexists. In contrast to the shared receptor, the latter receptor bindshIL13 but not hIL4. The restricted receptor is also sometimes called theglioma-associated receptor because it is preferentially expressed athigh levels in certain malignant cells, including high grade humangliomas. Debinski et al. (1995) Clin. Cancer Res., 1:1253-1258; andDebinski et al. (1996) J. Biol. Chem., 271, 22428-22433. In addition tobeing associated with malignancies, hIL13 has also been associated withother pathological conditions. Notably, IL13 has been shown to beinvolved in pathways that regulate airway inflammation, suggesting thatthis cytokine might play an important role in asthma and perhaps otherallergic pathologies. Webb et al., (2000) J. Immunol. 165:108-113; andDjukanovic, R. (2000) Clin. Exp. Allergy 30 Suppl 1:46-50.

SUMMARY OF THE INVENTION

The invention relates to the development and characterization of severalmutants of hIL13. Using these mutants, three regions of native hIL13were identified as being required for signaling through the sharedreceptor. These regions were localized to alpha-helices A, C and D andwere generally separated from the regions involved in binding to therestricted receptor. Glutamic acids at positions 13 and 16 in hIL13alpha-helix A, arginine and serine at positions 66 and 69 in helix C,and arginine at position 109 in helix D were found to be important ininducing biological signaling because these mutations resulted in theloss and/or gain of functional phenomena.

Mutants within the invention include those having one or more of thenative amino acids of hIL13 at positions 13, 16, 17, 66, 69, 99, 102,104, 105, 106, 107, 108, 109, 112, 113, and 114 replaced with adifferent amino acid. These mutants are expressed herein as hIL13X₁PX₂,where P is a number corresponding to the position of the mutated aminoacid in hIL13, X₁ is the letter abbreviation of the amino acid that wasreplaced, and X₂ is the letter abbreviation of the replacement aminoacid. For example, hIL13.E13K represents a mutant form of hIL13 that hasthe glutamic acid residue that naturally occurs at position 13 in nativehIL13 replaced with a lysine residue. Representative mutants within theinvention include hIL13.E13K, hIL13.E13I, hIL13.E13C, hIL13.E13S,hIL13.E13R, hIL13.E13Y, hIL13.E13D, hIL13.E16K, hIL13.E17K, hIL13.R66D,hIL13.S69D, hIL13.D99K, hIL13.L102A, hIL13.L104A, hIL13.K105D,hIL13.K106D, hIL13.L107A, hIL13.F108Y, hIL13.R109D, hIL13.R112D,hIL13.F113D, and hIL13.N114D.

Also within the invention are compositions including a mutant hIL13having an amino acid sequence having at least 90% sequence identity tonative hIL13 (SEQ ID NO:1). Such mutants can have a mutation in a domaincorresponding to the A, C, or D alpha-helices of native hIL13. Exemplarymutants include those with a polypeptide having an amino acid sequenceof one of SEQ ID NOs: 2-23.

Mutants of hIL13 within the invention can be those that specificallybind the shared IL4/IL13 receptor but not the restricted(IL4-independent) receptor; those that specifically bind the restricted(IL4-independent) receptor but not the shared IL4/IL13 receptor; orthose that bind both receptors.

Some hIL13 mutants of the invention specifically bind to an hIL13receptor associated with a cell in a manner that induces a measurablechange in the cell's physiology. This change can be of greater or lessmagnitude than a change in the cell's physiology that would be inducedby specifically binding the IL13 receptor with native hIL13.

Compositions within the invention can include both an hIL13 mutant and apharmaceutically acceptable carrier.

Mutants of hIL13 within the invention can be conjugated to an effectormolecule such as a cytotoxin (e.g., Pseudomonas exotoxin, PE38QQR, PE1E,PE4E, Diptheria toxin, ricin, abrin, saporin, and pokeweed viralprotein), a detectable label (e.g., radionuclide), an antibody, aliposome, or a lipid.

In another aspect the invention includes a purified nucleic acidencoding a mutant hIL13. Also within the invention is an antibody thatspecifically binds a mutant hIL13 molecule, but not a native hIL13molecule. And in another aspect, the invention features a method ofdelivering a mutant hIL13 to a cell. The method can include the stepsof: providing a mutant hIL13 and a cell; and contacting the cell withthe mutant hIL13.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Commonly understood definitions ofmolecular biology terms can be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

As used herein, the phrase “native hIL13” means the mature form of humaninterleukin 13, the amino acid sequence of which is shown herein as SEQID NO:1.

The phrase “hIL13 mutant” or a “mutant hIL13 molecule” means an hIL13 inwhich one or more of the amino acids differ from the corresponding aminoacids in the native hIL13. Thus, for example, where a native hIL13 has aglutamic acid at position 13, a mutant hIL13 can have an amino acidother than glutamic acid at position 13 (e.g., glutamic acid issubstituted with lysine). It will appreciated that mutant IL13 moleculesof this invention include mutant IL13 molecules of other mammalianspecies (e.g., rat, murine, porcine, ovine, goats, non-human primates,bovine, canus, and the like) and this invention contemplates the use ofmutant IL13 in veterinary as well as human medical conditions.

As used herein, the terms “protein” and “polypeptide” are usedsynonymously to mean any peptide-linked chain of amino acids, regardlessof length or post-translational modification, e.g., glycosylation orphosphorylation. An “purified” polypeptide is one that has beensubstantially separated or isolated away from other polypeptides in acell, organism, or mixture in which the polypeptide occurs (e.g., 30,40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid). A “purified” nucleic acid molecule is one thathas been substantially separated or isolated away from other nucleicacid sequences in a cell or organism in which the nucleic acid naturallyoccurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% freeof contaminants). The term includes, e.g., a recombinant nucleic acidmolecule incorporated into a vector, a plasmid, a virus, or a genome ofa prokaryote or eukaryote. Examples of purified nucleic acids includecDNAs, fragments of genomic nucleic acids, nucleic acids producedpolymerase chain reaction (PCR), nucleic acids formed by restrictionenzyme treatment of genomic nucleic acids, recombinant nucleic acids,and chemically synthesized nucleic acid molecules. A “recombinant”nucleic acid molecule is one made by an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

As used herein, “sequence identity” means the percentage of identicalsubunits at corresponding positions in two sequences when the twosequences are aligned to maximize subunit matching, i.e., taking intoaccount gaps and insertions. When a subunit position in both of the twosequences is occupied by the same monomeric subunit, e.g., if a givenposition is occupied by an alanine in each of two polypeptide molecules,then the molecules are identical at that position. For example, if 7positions in a sequence 10 amino acids in length are identical to thecorresponding positions in a second 10 amino acid sequence, then the twosequences have 70% sequence identity. Sequence identity is typicallymeasured using sequence analysis software (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705).

By the term “antibody” is meant an immunoglobulin as well as any portionor fragment of an immunoglobulin whether made by enzymatic digestion ofintact immunoglobulin or by techniques in molecular biology. The termalso refers to a mixture containing an immunoglobulin (or portion orfragment thereof) such as an antiserum.

The term “specifically binds”, as used herein, when referring to apolypeptide (including antibodies) or receptor, refers to a bindingreaction which is determinative of the presence of the protein orpolypeptide or receptor in a heterogeneous population of proteins andother biologics. Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody), the specified ligand or antibodybinds to its particular “target” (e.g. an IL13 specifically binds to anIL13 receptor) and does not bind in a significant amount to otherproteins present in the sample or to other proteins to which the ligandor antibody may come in contact in an organism. Generally, a firstmolecule that “specifically binds” a second molecule has a bindingaffinity greater than about 10⁵ (e.g., 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,and 10¹² or more) moles/liter for that second molecule.

A “mutation” in a polypeptide refers to the substitution of an aminoacid at a particular position in a polypeptide with a different aminoacid at that position. Thus, for example, the mutation hIL13.E13Kindicates that the native amino acid at position 13 in IL13 (glutamicacid, E) is replaced with lysine (K). In some cases, a mutation can bethe deletion, addition, or substitution of more than one amino acid in apolypeptide. The mutation does not require an actual removal andsubstitution of the amino acid(s) in question. The protein can becreated de novo with the replacement amino acid in the position(s) ofthe desired mutation(s) so the net result is equivalent to thereplacement of the amino acid in question.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions will control. Inaddition, the particular embodiments discussed below are illustrativeonly and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

FIG. 1 is a photograph of a SDS-PAGE (A) and Western blot (B) analysisof purified hIL13 and various hIL13 mutants. Five hundred nanograms ofeach purified cytokine was loaded per sample. Proteins were detectedusing a Coomassie Blue stain, panel A. Separated proteins from aduplicate gel were electroblotted to a PVDF membrane and detected withan anti-hIL13 antibody in a Western blot protocol using an enhancedchemiluminescence detection system, panel B.

FIG. 2 is circular dichroism (CD) spectra obtained from purified hIL13and various hIL13 mutants. Each protein was diluted in PBS (0.1 mg/ml),thermally equilibrated to 37L and its CD spectrum recorded over thewavelength range of 185 nm to 260 nm. The CD spectrum of unfolded hIL13(panel D) was obtained by diluting the protein in 8 M urea containing 40mM dithiothreitol prior to analysis. The reported spectra were theaverage of three consecutive measurements. The mutants in each panel,listed from top to bottom, represent the order of the spectra in eachpanel, from top to bottom.

FIG. 3 is graphical representations of data obtained from proliferationassays using TF-1 cells induced with hIL13 and various hIL13 mutants.TF-1 cells were cultured in the presence of increasing concentrations ofthe indicated protein for 72 h. The amount of TF-1 cell proliferation,compared to control experiments induced with buffer alone, wasdetermined colorimetrically. The reported data is the average oftriplicate samples with the error bars representing the standarddeviation within a data set. Experiments were repeated several times.Panels represents hIL13 aa-helix A mutants that increased TF-1 cellproliferation (A), hIL13 aa-helix A mutants that failed to increase TF-1cell proliferation (B), and hIL13 aa-helix C mutants that failed toincrease TF-1 cell proliferation (C).

FIGS. 4A-4J is a series of photomicrographs of indirectimmunofluorescence analyses of HUVEC for VCAM-1 expression induced byhIL13 and various hIL13 mutants. Panels A-F and G-J are from twoseparate experiments, each with its own set of controls. HUVEC cellswere cultured overnight in media containing buffer alone (panels A andG) or 1 mg/ml of either wild-type hIL13 (panels B and H) or variousmutants (panels C-F, I and J). Induced expression of the protein wasdetected through a rhodamine filter using goat anti-VCAM-1 IgG primaryantibody and rabbit anti-goat IgG CY3-conjugated secondary antibody. Thesensitivity of the imaging camera was set to detect the level offluorescence in the control field, panels A and G. No furtheradjustments were made to the sensitivity, allowing for the amount ofincreased or decreased fluorescence in the experimental fields to bedirectly related to the amount of interleukin-induced VCAM-1 expression.Photomicrographs are shown at 20× magnification (20×).

FIGS. 5A-5F are graphical representations of data obtained fromcytotoxicity assays performed to assess the ability of hIL13 and hIL13mutants to block the killing of U-251MG (FIGS. 5A and 5C) and SNB-19(FIGS. 5B and 5D) cells by hIL13-PE1E. Cultured cells were incubatedwith buffer alone, shown in all panels by closed diamonds, or 1 mg/ml ofhIL13 or the indicated mutant for 1 hour at 37° C. prior to the additionof increasing concentrations of hIL13-PE1E. The reported data is theaverage of triplicate samples with the error bars representing thestandard deviation within a data set. Experiments were repeated severaltimes.

DETAILED DESCRIPTION

This invention encompasses compositions and methods relating to hIL13mutants. The below described preferred embodiments illustrateadaptations of these compositions and methods. Nonetheless, from thedescription of these embodiments, other aspects of the invention can bemade and/or practiced based on the description provided below.

Biological Methods

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates). Varioustechniques using polymerase chain reaction (PCR) are described, e.g., inInnis et al., PCR Protocols: A Guide to Methods and Applications,Academic Press: San Diego, 1990. PCR-primer pairs can be derived fromknown sequences by known techniques such as using computer programsintended for that purpose (e.g., Primer, Version 0.5, ©1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). The ReverseTranscriptase Polymerase Chain Reaction (RT-PCR) method used to identifyand amplify certain polynuleotide sequences within the invention wasperformed as described in Elek et al., In Vivo, 14:172-182, 2000).Methods for chemical synthesis of nucleic acids are discussed, forexample, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981,and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemicalsynthesis of nucleic acids can be performed, for example, on commercialautomated oligonucleotide synthesizers. Immunological methods (e.g.,preparation of antigen-specific antibodies, immunoprecipitation, andimmunoblotting) are described, e.g., in Current Protocols in Immunology,ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods ofImmunological Analysis, ed. Masseyeff et al., John Wiley & Sons, NewYork, 1992.

Mutant hIL13 Molecules

The mutant hIL13 molecules of the invention are based on the amino acidsequence of native hIL13 (SEQ ID NO:1). The hIL13 mutants within theinvention differ by one or more amino acids from native hIL13. Forexample, hIL13 mutants within the invention can have 90% or more (e.g.,91, 92, 93, 94, 95, 96, 97, 98, and 99%) sequence identity with nativehIL13. Examples of hIL13 mutants within the invention are those havingthe amino acid sequences of SEQ ID NOs:2-23. These mutants each have amutation in a domain corresponding to either the A (residues 9-25 of SEQID NO:1), C (residues 59-71 of SEQ ID NO:1), or D (residues 97-113 ofSEQ ID NO:1) alpha-helices of native hIL13. Each of these features asubstitution of one of the amino acid residues that occurs in nativehIL13. Other hIL13 mutants within the invention are those with two ormore (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) such amino acidsubstitutions, as well as deletion (e.g., truncation) and addition(i.e., those with additional amino acids added to the native hIL13sequence) mutations.

Mutants of hIL13 can be made in a number of ways by adapting techniqueswell known in the art. See, e.g., Sambrook et al., supra; and Ausubel etal., supra. For example, starting with the known amino acid sequence ofhIL13 (i.e., SEQ ID NO:1), the skilled artisan can chemically synthesizevarious mutant hIL13 molecules using, e.g, automated commercialpolypeptide synthesizers. Techniques for solid phase synthesis ofpolypeptides are well known. See, e.g., Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984). Using this technique, hIL13 mutants can besynthesized as a single polypeptide. Alternatively, shorter oligopeptideportions of the mutant hIL13 molecule can first be synthesized and thenfused together to form the full length mutant by condensation of theamino terminus of one oligopeptide portion with the carboxyl terminus ofthe another oligopeptide portion to forming a peptide bond. The fusionscan then be purified by standard protein chemistry techniques.

Mutants of hIL13 can also be produced through recombinant expression ofhIL13-encoding nucleic acids (see below) in which the nucleic acid ismodified, randomly or in a site-specific manner, to change (substitute),add to, or delete, some or all of the amino acids in the encodedpolypeptide. Site-specific mutations can be introduced into theIL13-encoding nucleic acid by a variety of conventional techniques welldescribed in the scientific and patent literature. Illustrative examplesinclude: site-directed mutagenesis by overlap extension polymerase chainreaction (OE-PCR), as in Urban (1997) Nucleic Acids Res. 25: 2227-2228;Ke (1997) Nucleic Acids Res., 25: 3371-3372, and Chattopadhyay (1997)Biotechniques 22: 1054-1056, describing PCR-based site-directedmutagenesis “megaprimer” method; Bohnsack (1997) Mol. Biotechnol. 7:181-188; Ailenberg (1997) Biotechniques 22: 624-626, describingsite-directed mutagenesis using a PCR-based staggered re-annealingmethod without restriction enzymes; Nicolas (1997) Biotechniques 22:430-434, site-directed mutagenesis using long primer-unique siteelimination and exonuclease III. Unique-site elimination mutagenesis canalso be used (see, e.g., Dang et al. (1992) Anal. Biochem., 200: 81).The production of mutants of biologically active proteins such asIFN-beta and IL-2 is described in detail in U.S. Pat. No. 4,853,332 andthe mutation of hIL13 is described in Example 1 below.

Other hIL13 mutants can be prepared by chemically modifying native hIL13according to known chemical modification methods. See, e.g., Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896. Likewise,hIL13 mutants made by chemical synthesis or by expression of nucleicacids as described above can be chemically modified to make additionalhIL13 mutants.

Characterizing hIL13 Mutants

Mutants of hIL13 can have characteristics that differ from those nativehIL13. For example, native hIL13 has the functional characteristics ofbinding both shared receptor and the restrictive receptor. Native hIL13also has the characteristic of inducing transmembrane signals throughbinding shared receptors expressed on a cell surface. Such signaling canresult in a measurable change in the cell's physiology. Changes can bethe production of second messengers—e.g, an increase in intracellular[Ca²⁺], activation of protein kinases and/or phosphorylases, changes inphosphorylation of a substrate, changes in signal transducers andactivators of transcription, etc. They can also be changes in the cellproteome, e.g., from increased or decreased transcription ortranslation. Or they can be changes in a functional or phenotypiccharacteristic of the cell. For instance, adding native hIL13 to TF-1cells can increase their rate of proliferation. As another example,adding native hIL13 can cause HUVEC to increase their expression ofVCAM-1.

Characteristics of a given mutant hIL13 molecule can therefore beassessed by examining the ability of the molecule to bind the sharedreceptor and/or the restrictive receptor. Similarly, the ability of themutant molecule to induce transmembrane signaling can be assessed byexamining whether contacting a cell expressing an IL13 receptor with themutant molecule results in a change in the cell's physiology. By thesemethods, hIL13 mutants can be characterized as those that bind both theshared receptor and/or the restrictive receptor, those that bind onlyone of the receptors, and those that do not bind either receptor. Byquantifying the affinity of a mutant hIL13 molecule, it can also becharacterized as one that binds with less, about equal, or more affinitythan native hIL13. Mutants of hIL13 can also be characterized as havingor lacking the ability to cause a transmembrane signal and/or a changein a cell's function or phenotype. The changes caused by a mutant hIL13molecule can also be quantified to further characterize the molecule asone that causes such changes less than (of less magnitude), about equalto, or more than (of greater magnitude) those caused by native hIL13.For instance mutants of hIL13 that specifically bind to an hIL13receptor associated with a cell in a manner that induces a measurablechange in the cell's physiology can be those that modulate theproliferation rate of a cell line that expresses an IL13 receptor suchas TF-1 cells. Antagonistic hIL13 mutants are those that reduce theproliferation rate of the cell line compared to that induced by nativehIL13; agonistic hIL13 mutants are those that induce about the same(e.g., 50-150% or 75-125% of) proliferation rate of the cell line asthat induced by native hIL13; and superagonistic hIL13 mutants are thosethat increase the proliferation rate of the cell line compared to thatinduced by native hIL13. See Examples, below.

Chimeric Molecules of Mutant hIL13 and Effector Molecules

The invention also provides a chimeric molecule including a mutant hIL13molecule conjugated to an effector molecule. The effector molecule canbe any molecule that can be conjugated to an hIL13 mutant and exert aparticular function. Examples of effector molecules include cytotoxins,drugs, detectable labels, targeting ligands, and delivery vehicles.

A mutant hIL13 molecule conjugated with a one or more cytoxins can beused to kill cells expressing a receptor to which the mutant binds.Cytotoxins for use in the invention can be any cytotoxic agent (i.e.,molecule that can kill a cell after contacting the cell) that can beconjugated to hIL13 or an hIL13 mutant. Examples of cytotoxins include,without limitation, radionuclides (e.g., ³⁵S, ¹⁴C, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y,⁸⁹Zr, ²⁰¹Tl, ¹⁸⁶Re, ¹⁸⁸Re, ⁵⁷Cu, ²¹³Bi, ²¹¹At, etc.), conjugatedradionuclides, and chemotherapeutic agents. Further examples ofcytotoxins include, but are not limited to, antimetabolites (e.g.,5-flourouricil (5-FU), methotrexate (MTX), fludarabine, etc.),anti-microtubule agents (e.g., vincristine, vinblastine, colchicine,taxanes (such as paclitaxel and docetaxel), etc.), alkylating agents(e.g., cyclophasphamide, melphalan, bischloroethylnitrosurea (BCNU),etc.), platinum agents (e.g., cisplatin (also termed cDDP), carboplatin,oxaliplatin, JM-216, CI-973, etc.), anthracyclines (e.g., doxorubicin,daunorubicin, etc.), antibiotic agents (e.g., mitomycin-C),topoisomerase inhibitors (e.g., etoposide, tenoposide, andcamptothecins), or other cytotoxic agents such as ricin, diptheria toxin(DT), Pseudomonas exotoxin (PE) A, PE40, abrin, saporin, pokeweed viralprotein, ethidium bromide, glucocorticoid, and others. See, e.g. U.S.Pat. No. 5,932,188. Useful variations of PE and DT include PE38QQR (see,U.S. Pat. No. 5,614,191), PE1E and PE4E (see, e.g., Chaudhary et al.(1995) J. Biol. Chem., 265:16306), and DT388 and DT398 (Chaudhary, etal. (1991) Bioch. Biophys. Res. Comm., 180: 545-551) can also be used.

Mutant hIL13 molecules conjugated with one or more detectable labels canbe used to detect the presence of a receptor to which the mutant binds,e.g., in diagnostic assays (e.g., in the detection of shed tumor cellsoverexpression the IL13 receptor) and/or in the in vivo localization oftumor cells. Detectable labels for use in the invention can be anysubstance that can be conjugated to hIL13 or an hIL13 mutant anddetected. Suitable detectable labels are those that can be detected, forexample, by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful detectable labels in thepresent invention include biotin or streptavidin, magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, texasred, rhodamine, green fluorescent protein, and the like), radiolabels(e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, or ⁷²As,),radioopaque substances such as metals for radioimaging, paramagneticagents for magnetic resonance imaging, enzymes (e.g., horseradishperoxidase, alkaline phosphatase and others commonly used in an ELISA),and colorimetric labels such as colloidal gold or colored glass orplastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photo detector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label, and so forth.

Mutant hIL13 molecules conjugated with one or more targeting ligands(i.e., molecules that can bind a particular receptor) can be used tomediate binding of the mutants to a particular receptor or cellexpressing the receptor. Any targeting ligand that can be conjugated tohIL13 or an hIL13 mutant can be used. Examples of such targeting ligandsincludes antibodies (or the antigen-binding portion of antibodies); andchemokines, growth factors, soluble cytokine receptors (e.g., thoselacking a transmembrane domain), superantigens, or other molecules thatbind a particular receptor. A large number of these molecules are known,e.g., IL-2, IL-4, IL-6, IL-7, tumor necrosis factor (TNF), anti-Tac,TGF-alpha., SEA, SEB, and the like. As a representative example, anhIL13 mutant can be conjugated with a soluble form of a hIL13 receptor.This conjugate, for example, could be used to both antagonize anendogenous hIL13 receptor on a cell and neutralize any hIL13 present inthe vicinity of the cell.

Mutant hIL13 molecules conjugated with one or more nucleic acids can beused to specifically target delivery of the nucleic acid(s) to a targetcell (e.g., one expressing an receptor to which the mutant binds). Anynucleic acid that can be conjugated to hIL13 or an hIL13 mutant can beused. The nucleic acids can be attached directly to the mutant hIL13,attached via a linker, or complexed with or encapsulated in anothermoiety (e.g., a lipid, a liposome, a viral coat, or the like) that isattached to the mutant IL13 molecule. The nucleic acid can provide anyof number of effector functions. For example, a nucleic acid encodingone or more proteins can be used to deliver a particular enzymaticactivity, substrate, and/or epitope to a target cell. For theseapplications or others where expression (e.g. transcription ortranslation) of the nucleic acid is desired, the nucleic acid ispreferably a component of an expression cassette that includes all theregulatory sequences necessary to express the nucleic acid in the cell.Suitable expression cassettes typically include promoter initiation andtermination codons, and are selected to optimize expression in thetarget cell. Methods of constructing suitable expression cassettes arewell known to those of skill in the art. See, e.g., Sambrook et al.,supra.

A mutant hIL13 molecule conjugated with a one or more drugs can be usedto deliver such drug(s) to cells expressing a receptor to which themutant binds. Any drug which can be conjugated to hIL13 or an hIL13mutant can be used. Examples of such drugs include sensitizing agentsthat render a target (e.g., tumor) cell susceptible to various cancertherapeutics. The sensitizing agent can be a small molecule drug or agene (under the control of a promoter in an appropriate expressioncassette to induce expression in the target cell). For example, it hasbeen proposed that expression of the herpes simplex virus (HSV)thymidine kinase (TK) gene in proliferating cells, renders the cellssensitive to the deoxynucleoside analog, ganciclovir. Moolten et at.(1986) Cancer Res. 46:5276-5281; Moolten et al. (1990) Hum. Gene Ther.1: 125-134; Moolten et al. (1990) J. Natl. Cancer Inst. 82: 297-300;Short et al. (1990) J. Neurosci. Res. 27:427-433; Ezzedine et al. (1991)New Biol. 3: 608-614, Boviatsis et al. (1994) Hum. Gene Ther. 5:183-191. HSV-TK mediates the phosphorylation of ganciclovir, which isincorporated into DNA strands during DNA replication (S-phase) in thecell cycle, leading to chain termination and cell death. Elion (1983)Antimicr. Chemother. 12, sup. B:9-17. A second example of a gene with adrug-conditional “killing” function is the bacterial cytosine deaminasegene, which confers chemosensitivity to the relatively non-toxic5-fluorouracil precursor 5-fluorocytosine. Mullen et al. (1992) Proc.Natl. Acad. Sci. USA 89: 33-37; Huber et al. (1993) Cancer Res. 53:4619-4626; Mullen et al. (1994) Cancer Res. 54: 1503-1506. Still anotherexample of a gene with a drug-conditional “killing” function is acytochrome P450 gene. Expression, of the gene product renders tumorcells sensitive to a chemotherapeutic agent, in particular,cyclophosphamide or ifosphamide. See, U.S. Pat. No. 5,688,773. The drugemployed need not be a gene. For example, it can be one of the compoundsthat can treat multiple drug resistance of susceptible tumor cellsdescribed in U.S. Pat. No. 4,282,233. Other drugs can also be used. Forexample, chemotherapy drugs such as doxorubicin, vinblastine, genistein,and other described above can be conjugated to the mutant hIL13molecule.

A mutant hIL13 molecule conjugated to a one or more delivery vehicles isalso within the invention. Such conjugates can be used to deliver othersubstances such as a drug to cells expressing a receptor to which themutant binds. Any delivery vehicle that can be conjugated to hIL13 or anhIL13 mutant can be used. Examples of such delivery vehicles includeliposomes and lipids (e.g., micelles). Liposomes encapsulating drugs ormicelles including drugs may also be used. Methods for preparingliposomes attached to proteins are well known to those of skill in theart. See, for example, U.S. Pat. No. 4,957,735; and Connor et al.,Pharm. Ther., 28: 341-365 (1985).

Effector molecules can be conjugated (e.g., covalently bonded) to amutant hIL13 by any method known in the art for conjugating two suchmolecules together. For example, the mutant hIL13 can be chemicallyderivatized with an effector molecule either directly or using a linker(spacer). Several methods and reagents (e.g., cross-linkers) formediating this conjugation are known. See, e.g., catalog of PierceChemical Company; and Means and Feeney, Chemical Modification ofProteins, Holden-Day Inc., San Francisco, Calif. 1971. Variousprocedures and linker molecules for attaching various compoundsincluding radionuclide metal chelates, toxins, and drugs to proteins(e.g., to antibodies) are described, for example, in European PatentApplication No. 188,256; U.S. Pat. Nos. 4,671,958; 4,659,839; 4,414,148;4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al.Cancer Res. 47: 4071-4075 (1987). In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody- Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982); Waldmann (1991) Science, 252: 1657; and U.S.Pat. Nos. 4,545,985 and 4,894,443.

Where the effector molecule is a polypeptide, the chimeric moleculeincluding the hIL13 mutant and the effector can be a fusion protein.Fusion proteins can be prepared using conventional techniques inmolecular biology to join the two genes in frame into a single nucleicacid, and then expressing the nucleic acid in an appropriate host cellunder conditions in which the fusion protein is produced.

A mutant hIL13 may be conjugated to one or more effector molecule(s) invarious orientations. For example, the effector molecule may be joinedto either the amino or carboxy termini of the mutant hIL13. The mutantIL13 molecule may also be joined to an internal region of the effectormolecule, or conversely, the effector molecule may be joined to aninternal location of the mutant IL13 molecule.

In some circumstances, it is desirable to free the effector moleculefrom the mutant hIL13 molecule when the chimeric molecule has reachedits target site. Therefore, chimeric conjugates comprising linkages thatare cleavable in the vicinity of the target site may be used when theeffector is to be released at the target site. Cleaving of the linkageto release the effector molecule from the mutant IL13 molecule may beprompted by enzymatic activity or conditions to which the conjugate issubjected either inside the target cell or in the vicinity of the targetsite. When the target site is a tumor, a linker which is cleavable underconditions present at the tumor site (e.g. when exposed totumor-associated enzymes or acidic pH) may be used. A number ofdifferent cleavable linkers are known to those of skill in the art. See,e.g., U.S. Pat. Nos. 4,618,492; 4,542,225; and 4,625,014. The mechanismsfor release of an agent from these linker groups include, for example,irradiation of a photolabile bond and acid-catalyzed hydrolysis. U.S.Pat. No. 4,671,958, for example, includes a description ofimmunoconjugates comprising linkers which are cleaved at the target sitein vivo by the proteolytic enzymes of the patient's complement system.In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven effector molecule to a mutant hIL13 molecule.

Nucleic Acids Encoding Mutant hIL13 Molecules and Methods of MakingMutant hIL13 Molecules Using Nucleic Acids

The invention also provides purified nucleic acids encoding the mutanthIL13 molecules and the fusion proteins described above. Starting with aknown protein sequence, DNA encoding the mutant hIL13 molecules or thefusion proteins may be prepared by any suitable method, including, forexample, cloning and restriction of appropriate sequences or directchemical synthesis by methods such as the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.Because of the degeneracy of the genetic code, a large number ofdifferent nucleic acids will encode the mutant hIL13 molecules and thefusion proteins. Each of these is included within the invention.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. Longer DNA sequences may be obtained bythe ligation of shorter sequences. Alternatively, subsequences may becloned and the appropriate subsequences cleaved using appropriaterestriction enzymes. The fragments may then be ligated to produce thedesired DNA sequence.

DNA encoding the mutant hIL13 molecules or the fusion proteins may becloned using DNA amplification methods such as polymerase chain reaction(PCR). Thus, in a preferred embodiment, the gene for hIL13 is PCRamplified, using primers that introduce one or more mutations. Theprimers preferably include restrictions sites, e.g., a sense primercontaining the restriction site for NdeI and an antisense primercontaining the restriction site for HindIII. In one embodiment, theprimers are selected to amplify the nucleic acid starting at position19, as described by McKenzie et al. (1987), supra. This produces anucleic acid encoding the mature IL13 sequence (or mutant hIL13molecules) and having terminal restriction sites.

For making DNA encoding the fusion proteins, the DNA encoding theeffector molecule can be obtained from available sources. For example,the PE38QQR fragment may be excised from the plasmid pWDMH4-38QQR orplasmid pSGC242FdN1 as described by Debinski et al. Int. J. Cancer, 58:744-748 (1994), and by Debinski et al. ( 1994) Clin. Cancer Res. 1:1015-1022 respectively. Ligation of the mutant IL13 molecule and aPseudomonas exotoxin (e.g., PE38QQR) sequences and insertion into avector produces a vector encoding the mutant IL13 joined to the terminusof the Pseudomonas exotoxin (e.g., joined to the amino terminus ofPE38QQR, PE1E, or PE4E (position 253)). In a preferred embodiment, thetwo molecules are joined directly. Alternatively there can be anintervening peptide linker (e.g., a three amino acid junction consistingof glutamic acid, alanine, and phenylalanine introduced by therestriction site).

While the two molecules are preferably essentially directly joinedtogether, one of skill will appreciate that the molecules may beseparated by a peptide spacer consisting of one or more amino acids.Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the solubility, folding, net charge, or hydrophobicity.

The nucleic acid sequences encoding the mutant hIL13 molecules or thefusion proteins may be expressed in a variety of host cells, includingE. coli, other bacterial hosts, yeast, and various higher eukaryoticcells such as the COS, CHO and HeLa cells lines and myeloma cell lines.The recombinant protein gene will be operably linked to appropriateexpression control sequences for each host. F or E. coli this includes apromoter such as the T7, trp, or lambda promoters, a ribosome bindingsite and preferably a transcription termination signal. For eukaryoticcells, the control sequences will include a promoter and preferably anenhancer derived from immunoglobulin genes, SV 40, cytomegalovirus,etc., and a polyadenylation sequence, and may include splice donor andacceptor sequences.

Plasmid vectors of the invention made as described above can betransferred into the chosen host cell by well-known methods such ascalcium chloride, or heat shock, transformation for E. coli and calciumphosphate treatment or electroporation for mammalian cells. Cellstransformed by the plasmids can be selected by resistance to antibioticsconferred by genes contained on the plasmids, such as the amp, gpt, neoand hyg genes.

Once expressed, the recombinant mutant hIL13 molecules or fusionproteins can be purified according to standard procedures of the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like. See, generally, R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); andDeutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc. N.Y. (1990). Substantially purecompositions of at least about 90 to 95% homogeneity are preferred, and98 to 99% or more homogeneity are most preferred for pharmaceuticaluses. Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically.

After chemical synthesis, biological expression, or purification, themutant hIL13 molecules or the fusion proteins may possess a conformationsubstantially different than the native conformations of the constituentpolypeptides. In this case, it may be necessary to denature and reducethe polypeptide and then to cause the polypeptide to re-fold into thepreferred conformation. Methods of reducing and denaturing proteins andinducing re-folding are well known to those of skill in the art. See,Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al. (1992)Anal. Biochem., 205: 263-270.

Modifications can be made to the IL13 receptor targeted fusion proteinswithout diminishing their biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, amethionine added at the amino terminus to provide an initiation site, oradditional amino acids placed on either terminus to create convenientlylocated restriction sites or termination codons.

Antibodies

Mutants of hIL13 (or immunogenic fragments or analogs thereof) can beused to raise antibodies useful in the invention. Such polypeptides canbe produced by recombinant techniques or synthesized as described above.In general, hIL13 mutants can be coupled to a carrier protein, such asKLH, as described in Ausubel et al., supra, mixed with an adjuvant, andinjected into a host mammal. Antibodies produced in that animal can thenbe purified by peptide antigen affinity chromatography. In particular,various host animals can be immunized by injection with an hIL13 mutantor an antigenic fragment thereof. Commonly employed host animals includerabbits, mice, guinea pigs, and rats. Various adjuvants that can be usedto increase the immunological response depend on the host species andinclude Freund's adjuvant (complete and incomplete), mineral gels suchas aluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Other potentially useful adjuvantsinclude BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules that are contained in the sera of the immunized animals.Antibodies within the invention therefore include polyclonal antibodiesand, in addition, monoclonal antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, and molecules produced using a Fabexpression library. Monoclonal antibodies, which are homogeneouspopulations of antibodies to a particular antigen, can be prepared usingthe mutants of hIL13 described above and standard hybridoma technology(see, for example, Kohler et al., Nature 256:495, 1975; Kohler et al.,Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292,1976; Hammerling et al., “Monoclonal Antibodies and T Cell Hybridomas,”Elsevier, N.Y., 1981; Ausubel et al., supra). In particular, monoclonalantibodies can be obtained by any technique that provides for theproduction of antibody molecules by continuous cell lines in culturesuch as described in Kohler et al., Nature 256:495, 1975, and U.S. Pat.No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al.,Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA80:2026, 1983), and the EBV-hybridoma technique (Cole et al.,“Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp.77-96, 1983). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD and any subclass thereof. A hybridomaproducing a mAb of the invention may be cultivated in vitro or in vivo.The ability to produce high titers of mAbs in vivo makes this aparticularly useful method of production.

Once produced, polyclonal or monoclonal antibodies can be tested forspecific recognition of the mutants by Western blot orimmunoprecipitation analysis by standard methods, for example, asdescribed in Ausubel et al., supra. Antibodies that specificallyrecognize and bind to hIL13 mutants are useful in the invention. Forexample, such antibodies can be used to monitor the amount of an hIL13mutant associated with a cell or to block binding of a particular mutanta receptor.

Antibodies of the invention can be produced using fragments of the hIL13mutants that lie outside highly conserved regions and appear likely tobe antigenic, by criteria such as high frequency of charged residues.Cross-reactive anti-hIL13 mutant antibodies are produced using afragment of a hIL13 mutant that is conserved amongst members of thisfamily of proteins. In one specific example, such fragments aregenerated by standard techniques of PCR, and are then cloned into thepGEX expression vector (Ausubel et al., supra). Fusion proteins areexpressed in E.coli and purified using a glutathione agarose affinitymatrix as described in Ausubel, et al., supra. Non-cross reactiveantibodies can be prepared by adsorbing the antibody with the antigen(s)that the antibody is desired not to react with. For example, antiseraprepared against a particular hIL13 mutant can be adsorbed with otherhIL13 mutants and/or native hIL13 to reduce or eliminatecross-reactivity.

In some cases it may be desirable to minimize the potential problems oflow affinity or specificity of antisera. In such circumstances, two orthree fusions can be generated for each protein, and each fusion can beinjected into at least two rabbits. Antisera can be raised by injectionsin a series, preferably including at least three booster injections.Antiserum is also checked for its ability to immunoprecipitaterecombinant mutants of hIL13 or control proteins, such as glucocorticoidreceptor, CAT, or luciferase.

Techniques described for the production of single chain antibodies (U.S.Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can be adapted to producesingle chain antibodies against an hIL13 mutant, or a fragment thereof.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

The antibodies of the invention can be used, for example, in thedetection of a hIL13 mutant in a biological sample. Antibodies can alsobe used to interfere with the interaction of an hIL13 mutant and othermolecules that bind the mutant (e.g., an hIL13 receptor).

Methods of Delivering a Mutant hIL13 Molecule to a Cell

The invention also provides a method of delivering an IL13 mutant to acell. This method is useful, among other things, for directing achimeric molecule including the hIL13 mutant and an effector molecule toa cell so that the effector molecule can exert its function. Forexample, an hIL13 mutant conjugated to a cytotoxin can be delivered to atarget cell to be killed by mixing a composition containing the chimericmolecule with the target cell expressing a receptor that binds themutant. As another example, an hIL13 mutant conjugated to a detectablelabel can be directed to a target cell to be labeled by mixing acomposition containing the chimeric molecule with the target cellexpressing a receptor that binds the mutant.

Mutant hIL13 molecules can be delivered to a cell by any known method.For example, a composition containing the hIL13 mutant can be added tocells suspended in medium. Alternatively, a mutant hIL13 can beadministered to an animal (e.g., by a parenteral route) having a cellexpressing a receptor that binds the mutant so that the mutant binds tothe cell in situ. The mutant ILI3 molecules of this invention areparticularly well suited as targeting moieties for binding tumor cellsbecause tumor cells overexpress ILI3 receptors. In particular, carcinomatumor cells (e.g. renal carcinoma cells) overexpress ILI3 receptors atlevels ranging from about 2100 sites/cell to greater than 150,000 sitesper cell. Similarly, gliomas and other transformed cells alsooverexpress ILI3 receptors (ILI3R). Thus, the methods of this inventioncan be used to target an effector molecule to a variety of cancers. Suchcancers are well known to those of skill in the art and include, but arenot limited to, cancers of the skin (e.g., basal or squamous cellcarcinoma, melanoma, Kaposi's sarcoma, etc.), cancers of thereproductive system (e.g., testicular, ovarian, cervical), cancers ofthe gastrointestinal tract (e.g., stomach, small intestine, largeintestine, colorectal, etc.), cancers of the mouth and throat (e.g.esophageal, larynx, oropharynx, nasopharynx, oral, etc.), cancers of thehead and neck, bone cancers, breast cancers, liver cancers, prostatecancers (e.g., prostate carcinoma), thyroid cancers, heart cancers,retinal cancers (e.g., melanoma), kidney cancers, lung cancers (e.g.,mesothelioma), pancreatic cancers, brain cancers (e.g. gliomas,medulloblastomas, meningiomas, etc.) and cancers of the lymph system(e.g. lymphoma). In a particularly preferred embodiment, the methods ofthis invention are used to target effector molecules to brain cancers(especially gliomas).

One of skill in the art will appreciate that identification andconfirmation of ILI3 overexpression by other cells requires only routinescreening using well-known methods. Typically this involves providing alabeled molecule that specifically binds to the ILI3 receptor (e.g., anative or mutant ILI3). The cells in question are then contacted withthis molecule and washed. Quantifying the amount of label remainingassociated with the test cell provides a measure of the amount of ILI3receptor (ILI3R) present on the surface of that cell. In a preferredembodiment, IL13 receptor may be quantified by measuring the binding of¹²⁵-labeled IL13 (¹²⁵I-ILI3) to the cell in question. Details of such abinding assay are provided in U.S. Pat. No. 5,614,191.

As IL13 has been implicated in playing an important regulatory role inallergic hyperactivity reactions such as asthma (Webb et al. (2000) J.Immunol. 165:108-113), the invention also provides a method ofmodulating an allergic response by contacting a cell important in theresponse (e.g., a lymphocyte such as a B cell, an eosinophil, a mastcell, and/or any other cells involved in Th₂-dominated inflammatoryresponses) with one or more hIL13 mutants. Thus, for example, whereinteraction of native hIL13 with an hIL13 receptor expressed on a cellcauses transmembrane signals that contribute to the cell's role in anallergic reaction (e.g., inducing inflammation), a mutant hIL13 can beused to block this interaction and inhibit the allergic reaction. Theinteraction between native hIL13 and the IL13 receptor can be blocked,for example, by contacting the cell 1 can with an hIL13 mutant thatbinds to the IL13 receptor (in some cases with more affinity than nativehIL13) but does not cause the transmembrane signaling through thereceptor. For asthma, such an hIL13 mutant could be administered byinhalation of a pharmaceutical composition containing the mutant.

Pharmaceutical Compositions The mutant hIL13 molecules (including thoseconjugated with an effector molcule) of this invention can be preparedfor parenteral, topical, oral, or local administration, such as byaerosol or transdermally, for prophylactic and/or therapeutic treatment.The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include powder,tablets, pills, capsules and lozenges. It some cases it may be desirableto protect the fusion proteins and pharmaceutical compositions of thisinvention, from being digested (e.g., when administered orally). Thiscan be accomplished either by complexing the protein with a compositionthat renders it resistant to acidic and enzymatic hydrolysis, or bypackaging the protein in an appropriately resistant carrier such as aliposome. Means of protecting compounds from digestion are well known inthe art (see, e.g., U.S. Pat. No. 5,391,377 describing lipidcompositions for oral delivery of therapeutic agents).

The pharmaceutical compositions can also be delivered to an animal byinhalation by any presently known suitable technique. For example, thehIL13 mutants of the invention can be delivered in the form of anaerosol spray produced from pressurized packs or a nebulizer, with theuse of a suitable propellant such as dichlorodifluromethane,trichlorotri-fluoromethane, dichlorotetraflurorethane, carbon dioxide,or any other suitable gas. In the case of a pressurized aerosol, thedosage unit may be controlled using a valve to deliver a metered amount.Capsules and cartridges (e.g., of gelatin) containing a powder mix ofthe hIL13 mutant and a suitable base (e.g., lactose or starch) can beused in an inhaler or insufflator to deliver the mutant to therespiratory tract of an animal.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a solution of themutant hIL13 molecule dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter (e.g., pyrogens). Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of the mutant hIL13 in these formulations can vary widely,and will be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs. Actual methods forpreparing parenterally administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980).

Toxicity and therapeutic efficacy of the pharmaceutical compositionsutilized in the invention can be determined by standard pharmaceuticalprocedures, using either cells in culture or experimental animals todetermine the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Doses that exhibitlarge therapeutic indices are preferred. While those that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets the pharmaceutical composition to the site ofaffected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch pharmaceutical compositions lies preferably within a range ofcirculating concentrations that include an ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anypharmaceutical composition used in a method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve anIC₅₀ (that is, the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography. Although dosage should be determinedfor each particular application, it is expected that a dose of a typicalpharmaceutical composition for intravenous administration would be about0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mgper patient per day may be used, particularly when the pharmaceuticalcompositions is administered to a secluded site and not into the bloodstream, such as into a body cavity or into a lumen of an organ.

The compositions containing the present hIL13 mutants, or a cocktailthereof (i.e., with other proteins), can be administered for therapeutictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease, in an amount sufficient to cureor at least partially arrest the disease and its complications. Anamount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend upon theseverity of the disease and the general state of the patient's health.Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

Among various uses of the cytotoxic fusion proteins of the presentinvention are included a variety of disease conditions caused byspecific human cells that may be eliminated by the toxic action of theprotein. One preferred application is the treatment of cancer (e.g., aglioma), such as by the use of an mutant IL13 ligand attached to acytotoxin (e.g., PE or a PE derivative).

It will be appreciated by one of skill in the art that there are someregions that are not heavily vascularized or that are protected by cellsjoined by tight junctions and/or active transport mechanisms whichreduce or prevent the entry of macromolecules present in the bloodstream. For example, systemic administration of therapeutics to treatgliomas, or other brain cancers, is constrained by the blood-brainbarrier which resists the entry of macro-molecules into the subarachnoidspace. Thus, the therapeutic compositions of this invention can beadministered directly to the tumor site. For instance, brain tumors(e.g., gliomas) can be treated by administering the therapeuticcomposition directly to the tumor site (e.g., through a surgicallyimplanted catheter). Where the fluid delivery through the catheter ispressurized, small molecules ( e.g. the therapeutic molecules of thisinvention) will typically infiltrate as much as two to three centimetersbeyond the tumor margin.

Alternatively, the therapeutic composition can be placed at the targetsite in a slow release formulation (e.g., a thrombin-fibrinogenmixture). Such formulations can include, for example, a biocompatiblesponge or other inert or resorbable matrix material impregnated with thetherapeutic composition, slow dissolving time release capsules ormicrocapsules, and the like.

Typically the catheter, or catheters, or time release formulation willbe placed at the tumor site as part of a surgical procedure. Thus, forexample, where major tumor mass is surgically debulked, the perfusingcatheter or time release formulation can be emplaced at the tumor siteas an adjunct therapy. Of course, surgical removal of the tumor mass maybe undesired, not required, or impossible, in which case, the deliveryof the therapeutic compositions of this invention may comprise theprimary therapeutic modality.

Imaging

The invention also provides a method of imaging a cell expressing areceptor that binds an hIL13 mutant in vivo. In an exemplary method, anhIL13 mutant conjugated to a label detectable by the chosen imagingtechnique is administered to an animal having the cell expressing areceptor that binds the particular hIL13 mutant. The animal is thenimaged using the chosen imaging technique. Examples of labels useful fordiagnostic imaging include radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I, ⁹⁹mTc,³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh; fluorescent labels such as fluoresceinand rhodamine; nuclear magnetic resonance active labels; positronemitting isotopes detectable by a positron emission tomography (“PET”)scanner; chemiluminescent labels such as luciferin; and enzymaticmarkers such as peroxidase or phosphatase. Mutants of hIL13 can belabeled with such reagents as described above or using techniques knownin the art.

Any imaging technique compatible with the labeled-hIL13 mutant can beused. Examples of such techniques include immunoscintigraphy where agamma camera is used to detect the location and distribution ofgamma-emitting radioisotopes; MRI where a paramagnetic labeled-hIL13mutant is used; PET where an hIL13 mutant is conjugated with a positronemitting label; and X-ray imaging where an hIL13 mutant is conjugatedwith a radioopaque label (e.g., a metal particle). A more detaileddescription of such techniques is provided in Handbook of TargetedDelivery of Imaging Agents (Handbook of Pharmacology and Toxicology),ed. V. Torchilin, CRC Press, 1995; Armstrong et al., Diagnostic Imaging,Blackwell Science Inc., 1998; and Diagnostic Nuclear Medicine, ed. C.Schiepers, Springer Verlag, 2000.

As an illustrative example, the location of glioma tumor cells in ananimal can be determined by injecting (e.g., parenterally or in situ) ananimal with a composition including native hIL13 or an hIL13 mutantconjugated to a detectable label (e.g., a gamma emitting radioisotope).The composition is then allowed to equilibrate in the animal, and tobind to the glioma cells. The animal is then subjected to imaging (e.g.,using a gamma camera) to image where the glioma cells are.

Diagnostic Kits

In another embodiment, this invention provides for kits for thetreatment of tumors or for the detection of cells overexpressing IL 13receptors. Kits will typically comprise a chimeric molecule of thepresent invention (e.g., a mutant hIL13 conjugated to a detectablelabel, a mutant hIL13 conjugated to cytotoxin, a mutant IL13 conjugatedto a targeting ligand, etc.). In addition the kits will typicallyinclude instructional materials disclosing means of use of chimericmolecule ( e.g., as a cytotoxin, for detection of tumor cells, toaugment an immune response, etc.). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, where a kit contains a chimeric molecule inwhich the effector molecule is a detectable label, the kit mayadditionally contain means of detecting the label (e.g. enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a sheep anti-mouse-HRP, orthe like). The kits may additionally include buffers and other reagentsroutinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

EXAMPLES

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and are not tobe construed as limiting the scope or content of the invention in anyway.

Example 1 Materials and Methods

Restriction endonucleases and DNA ligase were obtained from New EnglandBiolabs (Beverly, Mass.), Bethesda Research Laboratories (BRL,Gaithersburg, Md.) and Boehringer Mannheim (Indianapolis, Ind.). U.S.E.mutagenesis kit, fast protein liquid chromatographic (FPLC) system,columns and media were obtained from Pharmacia (Piscataway, N.J.).Oligonucleotide primers were synthesized at the Macromolecular CoreLaboratory, Penn State College of Medicine. Polymerase chain reaction(PCR) kit was from Perkin-Elmer Cetus (Norwalk, Conn.). Tissue cultureware was from Corning (Corning, N.Y.).3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(inner salt)/phenazine methasulfate (MTS/PMS) non-radioactive cellproliferation assay was purchased from Promega (Madison, Wis.). SDS-PAGEsupplies were from BioRad (Hercules, Calif.). Antibodies were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, Calif.). SuperSignalSubstrate for chemiluminescent detection was purchased from Pierce(Rockford, Ill.). Cell lines were obtained from the American TypeCulture Collection (Rockville, Md.). MTS/PMS for cell titer 96 aqueousnon-radioactive cell proliferation assay was purchased from Promega(Madison, Wis.).

For recombinant protein expression in a prokaryotic system, all plasmidscarrying the genes encoding proteins of interest were under a T7promoter-based expression system. The plasmids were constructed asdescribed in Debinski et al., (1998) Nature Biotech., 16:449-453.BL21(□) E. coli, which carry the T7 RNA polymerase gene in anisopropyl-1-thio-blactopyranoside (IPTG) inducible form, were used asthe host for recombinant protein expression. Production of recombinantproteins driven by T7 RNA polymerase allowed production of milligramquantities of recombinant protein from a 1.0 liter culture induced atA₆₀₀ of 2.0.

For expression of proteins, competent BL21 cells were transformed withthe appropriate plasmids and grown in Terrific Broth (DIFCOLaboratories, Detroit Mich.) to A₆₀₀ equal to 2.0, at which point IPTGwas added to a final concentration of 250 Cells were harvested 90 min.later. The inclusion body fraction of the cells was isolated anddenatured in 7M guanidine HCl, then renatured by rapid dilution intobuffer, using the disulfide-shuffling method as was previously describedin Debinski et al. (1993) J. Biol. Chem., 268:14065-14070. Afterdialysis, the renatured proteins were purified using a Pharmacia fastprotein liquid chromatography (FPLC) system.

For mutagenesis, mutations of the hIL13 gene were made by standard PCRprotocols (using the mutated oligonucleotides as sense or anti-senseprimers in PCR) or by using a unique site elimination (U.S.E.)mutagenesis kit, based on the procedure developed by Deng and Nickoloffin Anal. Biochem., 200:81-88, 1992. Examples of primers used for themutagenesis are shown below in table 1. All mutated plasmids wereisolated and sequenced to verify the correct mutation prior to use.TABLE 1 hIL13.E13D: TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTC (SEQ ID NO: 8)CCTCTACAGCCCTCAGGGACCTCATTGAGGAG hIL13.E13I:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTC (SEQ ID NO: 3)CCTCTACAGCCCTCAGGATCCTCATTGAGGAG hIL13.E13K:AGGAGATATACATATGTCCCCAGGCCCTGTGCCT (SEQ ID NO: 2)CCCTCTACAGCCCTCAGGAAGCTCATTGAGGA hIL13.E13R:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTC (SEQ ID NO: 6)CCTCTACAGCCCTCAGGCGCCTCATTGAGGAG hIL13.E13S:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTC (SEQ ID NO: 5)CCTCTACAGCCCTCAGGTCTCTCATTGAGGAG hIL13.E13Y:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTC (SEQ ID NO: 7)CCTCTACAGCCCTCAGGTACCTCATTGAGGAG hIL13.E16K:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTC (SEQ ID NO: 9)CCTCTACAGCCCTCAGGGAGCTCATTAAGGAGCT GGT hIL13.E17K:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTC (SEQ ID NO: 10)CTCTACAGCCCTCAGGGAGCTCATTGAGAAGCTG GTCA hIL13.R66D:ATCGAGAAGACCCAGGACATGCTGAGCGGATTC (SEQ ID NO: 11) hIL13.[D]S69D:ACCCAGAGGATGCTGGACGGATTCTGCCCGCAC (SEQ ID NO: 12)

For polyacrylamide gel electrophoresis and immunoblotting, the purity ofthe isolated recombinant proteins was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, under nonreducingconditions. The separated proteins in the gel were stained either withCoomassie Blue for visual inspection or transferred to polyvinylidenedifluoride (PVDF) membrane for Western blot analysis. For Western blotanalysis, the PVDF with the transferred proteins was incubated in 5%nonfat milk in phosphate buffered saline (PBS) for one hour at roomtemperature. The membrane was incubated for one hour in 5% milk/PBScontaining goat anti-human IL13 antibody (1:1,000 dilution). Theantibody was raised against a hIL13 specific peptide located at thecarboxy terminus of hIL13. After incubation with the primary antibody,the membrane was washed three times, five min. each, with 0.05% Tween20/PBS. The membrane was then incubated for one hour in 5% milk/PBScontaining donkey anti-goat IgG conjugated with horseradish peroxidase(1:20,000 dilution). The membrane was washed three times, five min.each, with 0.05% Tween 20/PBS. The immuno-reactive proteins wereidentified on film, using enhanced chemiluminescence detection. Imageswere digitized using a Hewlett Packard Scan-Jet 6100C scanner andcomposited using Microsoft Powerpoint software.

For circular dichroism (CD), CD spectra for the proteins were obtainedover the wavelength range of 185-260 nm using a Jasco J-710spectropolarimeter. All measurements were carried at 37 Lusing the samecuvette, the same orientation of the cuvette to the light source, and a2 mm light path. Proteins (0.1 mg/ml) were resuspended in phosphatebuffered saline (PBS) and then analyzed. For unfolded samples, proteinwas resuspended in 8M urea containing 40 mM DTT (denaturation buffer).Reported spectra were the average of three consecutive runs for eachsample. Spectra from appropriate blanks, PBS alone or denaturationbuffer, were subtracted from each sample so that the resulting spectrareflected only the CD contribution of the proteins.

For cell proliferation assays, cell killing by cytotoxins was tested asfollows. 5×10³ cells per well were plated in a 96-well tissue cultureplate in 150 f media. Various concentrations of cytotoxins were dilutedin 0.1% BSA/PBS and 25 f each dilution was added to cells 18-24 hfollowing cell plating. Cells were incubated at 37° C. for another 48 h.Then, the cytotoxicity was determined using a calorimetric MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt]/PMS (phenazine methasulfate) cell proliferation assay.MTS/PMS was added at a half final concentration as recommended by themanufacturer. The cells were incubated with the dye for 4 hr and thenthe absorbance was measured at 490 nm for each well using a micro-platereader (Cambridge Technology, Inc., Watertown, Mass.). The wellscontaining cells treated with cycloheximide (10 mM) or wells with noviable cells left served as a background for the assay. For blockingstudies, interleukins at a concetration of 1.0 ug/ml were added to cellsfor 60 min before the cytotoxins addition.

Cell proliferation studies using TF-1 cells (pre-leukemic human B cells,which express the shared IL13/4 receptor) were performed by growing thecells in the presence of different concentrations of wild-typeinterleukins or their mutants in 96 well culture plates. After 72 h ofincubation at 37 L the rate of proliferation of the TF-1 cells wasdetermined by a calorimetric MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt]/PMS (phenazine methasulfate) cell proliferation assay. Thecell samples were incubated with the dye for four h then theirabsorbance at 490 nm was recorded for each well using a microplatereader. The wells with cells treated with high concentrations ofcycloheximide served as background for the assay.

For indirect immunofluorescence analyses, HUVEC were seeded onto aneight chambered slide, 50,000 cells per chamber, and incubated overnightat 37 Lo allow cells to attach. The media was removed and replaced withmedia containing hIL13 or its mutants (1 l final concentration). Thecells were incubated again overnight at 37 L The next day, the media wasremoved, the cells were fixed in ethanol and incubated with blockingmedia (10% normal rabbit serum in PBS) at room temperature for 20 min.The blocking media was removed and goat anti-VCAM-1 antibody (1 ug/ml)in 1.5% normal rabbit serum/PBS was added. Cells were incubated at roomtemperature for one hour, then primary antibody was removed and cellsrinsed three times, five min. each, with PBS. Cells were incubated withrabbit anti-goat IgG-Cy3 conjugate (1:150 dilution) in 1.5% normalrabbit serum/PBS for 45 min. at room temperature, in the dark. After 45min., the cells were rinsed three times, five min. each, with PBS, acoverslip was mounted using aqueous mounting medium, and the fluorescentstaining determined using a rhodamine filter set. Images were obtainedfrom the same experiment without adjusting the microscope betweensamples on a Zeiss Axioplan microscope and captured digitally usingSnappy by Play Inc.

For cytotoxicity-blocking assays, glioblastoma cells (U-251 MG andSNB-19) were plated into 96-well culture plates and incubated for 24 h.After 24 h, hIL13 or its mutants were added to cells and incubated forone hour at 37 L An equal volume of 0.1% BSA in PBS was added to cellsfor assays without blocking ligand. After the hour incubation,increasing concentration of the hIL13 chimeric toxin (hIL13-PE1E; seeDebinski, et al. (1996) J. Biol. Chem., 271, 22428-22433)) was added(0.001-10 ng/ml final concentration) and the cells were incubated forthree days. After three days, the number of proliferating cells in eachwell was determined using the colorimetric MTS/PMS method describedabove. The wells with cells treated with high concentrations ofcycloheximide served as background for the assay.

For autoradiography, recombinant hIL13.E13Y was iodo-labeled with ¹²⁵Iby using the IODO-GEN reagent (Pierce) according to the manufacturer'sinstructions. The specific activity of ¹²⁵I-hIL13.E13Y was ˜300 fprotein. All studies involving human specimens were approved by therespective Human Subjects Protection Offices at the Penn State Collegeof Medicine (Protocol No. IRB 96-123EP). Serial tissue sections were cut(10 on a cryostat, thaw-mounchrome alumme-alum coated slides, and storedat 4° C. until analyzed. To observe binding distribution of¹²⁵I-hIL13.E13Y, sections were incubated (1 hr, 22° C.) with 1.0 nM¹²⁵I-hIL13 in binding buffer (200 mM sucrose, 50 mM HEPES, 1% BSA, 10 mMEDTA). Adjacent serial sections were incubated with the radiolabeledrecombinant hIL13.E 13Y after a 30 min pre-incubation at 22° C. in thepresence of binding buffer alone or of a 100- to 500-fold molar excessof unlabeled hIL13, hIL13.E13Y or hIL4, or a monoclonal antibody againsthuman transferrin receptor (TfR). To dissociate non-specifically boundradioligand, sections were rinsed in four consecutive changes (5 minuteseach) of ice-cold 0.1 M PBS. At least two sections of each of the tissuespecimens were assayed for the evaluation of ¹²⁵I-hIL13.E13Y bindingspecificity. After drying, labeled sections were apposed to Kodakautoradiography film at −65° C. for 8 hr to 11 days.

Example 2 Radioimmunodetection and Radioimmunotherapy of Human HighGrade Gliomas

The IL13 mutein, hIL13.E13Y, was prepared as described above and testedfor its ability to modulate the interleukin-induced proliferativeresponses of TF-1 cells. TF-1 cells were treated with hIL13, hIL13.E13Y,or hIL13.E13K. While hIL13 was very potent in stimulating the growth ofTF-1 cells, hIL13.E13K showed no activity and hIL13.E13Y exhibited onlyvery weak activity, if any at all.

The ability of hIL13.E13Y to compete for the hIL13 binding sites inclinical specimens of glioblastoma (GBM) in situ was investigated inautoradiographic studies. The two GBM tissues studied labeled denselywith ¹²⁵I-hIL13.E13Y binding sites, as well as with labeled wild typehIL13. The binding was specific since both hIL13.E13Y and the wild typeIL13 blocked the binding of ¹²⁵1-hIL13.E13Y. In contrast, an excess ofrecombinant hIL4 was largely without influence on the ¹²⁵I-hIL13.E13Ybinding to GBM specimens.

In another test of specificity of the hIL13.E13Y binding to GBM, theability of a monoclonal antibody against the transferrin receptor (TfR)to displace the binding of radiolabeled interleukin was examined. Nocross-competition for the hIL13 binding sites in the GBMs examined wasobserved. The binding of hIL13.E13Y to GBM appears to be very specificas others studies have shown that ¹²⁵I-hIL13 fails to interact withnormal brain or normal human cells and that ¹²⁵I -hIL13.E13Y does notinteract with normal human cells, such as HUVEC.

In other tests, the ability of hIL13.E13Y to block the action ofhIL13-PE1E (an hIL13-based cytotoxin) was investigated using twodifferent human malignant glioma cell lines. Glioma cells in culturewere pretreated with either hIL13, hIL13.E13Y or hIL13.E13K beforehIL13-PE1E was added. The cytotoxicity of hIL13-PE1E was neutralized inthese cultures using hIL13, hIL13.E13Y, or hIL13.E13K.

Example 3 Mutants of Interleukin 13 with Altered Reactivity TowardsInterleukin 13 Receptors

Recombinant IL-13 and IL-13 mutants were prepared, isolated, andpurified as described in Example 1. The prokaryotic production of thecytokines or their mutants under control of the T7 promoter was veryefficient. After purification, between 0.5 mg and 1.5 mg of eachcytokine or mutant was obtained from a 1 liter culture. When eachpurified protein was analyzed using SDS-PAGE and stained with CoomassieBlue, a single protein band was observed migrating at approximately 13kDa (FIG. 1, panel A). Visual inspection suggested that all preparationswere greater than 95% pure. A corresponding Western blot of the samplesusing a goat polyclonal anti-hIL13 antibody (that was notcross-reactivity with any other cytokine) indicated that the isolatedproteins were immuno-reactive with hIL13 (FIG. 1, panel B). The aa-helixD mutants, hIL13.R109D and hIL13.F113D, also reacted with this antibodyindicating that they too were immuno-reactive with hIL13 (data notshown). Traces of a dimeric form (˜26 kDa) of some of the mutatedcytokines were also detected.

To determine whether the recombinant interleukins had refolded correctlyand that their mutation had not destroyed their general pattern ofconformation, circular dichroism (CD) was used to determine theproteins' folded structure. The secondary structure data from thespectropolarimeter indicated that each protein sample produced aspectrum consistent with an aa-helical enriched protein, having twospectral minima at approximately 208 nm and 222 nm (FIG. 2).Furthermore, the CD spectrum of each mutant could be super-imposed onthe CD spectrum of the wild-type hIL13, although slight variations inspectra intensity were observed between samples (FIG. 2, panels A, B,C). hIL13.R109D and hIL13.F113D both produced CD spectra similar to theother mutants (not shown). For comparison, the CD spectrum of unfoldedhIL13 was also obtained (FIG. 2, panel D). The panel illustrates thecollapse of the characteristic alpha-helical pattern when the protein isunfolded.

Functional assays were employed to examine whether the IL13 mutantsexhibited an altered association with the shared signaling IL13/4receptor by measuring their effect on induced TF-1 cell proliferation.TF-1 cells express the shared IL13/4 receptor (but not the restrictedreceptor) and proliferate in a dose-dependent manner in the presence ofhIL13 or hIL4. Under the conditions used in this assay, a concentrationof 100 ng/ml of wild-type hIL13 consistently produced a maximalproliferative response in TF-1 cells of ˜300% that of the baseline value(FIG. 3, panel A). Differences were observed in TF-1 cell proliferationdepending on whether the mutants were in the predicted aa-helices A, C,or D. Of the aa-helix A mutants, hIL13.E13K induced only a minimalproliferative response over the range tested (FIG. 3, panel B), andhIL13.E13I, hIL13.E13S, and hIL13.E13Y failed to induce anyproliferative response (FIG. 3, panel B). Mutants hIL13.E13D and,unexpectedly, hIL13.E13R both induced a dose-dependent increase inproliferation of the TF-1 cells. Their induction of TF-1 cellproliferation followed the same pattern as wild-type hIL13, althoughhIL13.E13D had a lesser effect on proliferation than did hIL13.E13R(FIG. 3, panel A). Both hIL13.E16K and hIL13.E17K (with mutated sitesone turn of the aa-helix up from position 13) induced a dose-dependentincrease in the proliferative response of the TF-1 cells (FIG. 3, panelA). While the hIL13.E17K-induced effect was comparable to wild-typehIL13, the hIL13.E16K-induced effect was significantly greater than thatcaused by wild-type hIL13.

The aa-helix C mutants, hIL13.R66D and hIL13.S69D, both showed asignificantly impaired ability to stimulate TF-1 cells, compared towild-type hIL13 (FIG. 3, panel C) Their action on TF-1 cells, however,can be classified between that caused by mutants shown in FIG. 3, panelsA and B. The aa-helix D mutants also exhibited contrasting patterns ofaction on TF-1 cells. The hIL 13.F113D mutant was equivalent towild-type hIL13 in inducing TF-1 cell proliferation, while thehIL13.R109D mutant was inactive on these cells (not shown).

The ability of the hIL13 mutants to interact with the shared hIL13/4receptor on normal cells was assessed by examining their effect onVCAM-1 expression on the surface of HUVEC. Cytokine binding of theshared IL13/4 receptor on the HUVEC cell surface results intransmembrane signaling events that induce VCAM-1 expression on thesecells. Results from two separate experiments are shown in FIG. 4. Cellsincubated in the absence of hIL13 showed minimal, nonspecific VCAM-1staining (FIG. 4, panels A and G). In contrast, cells incubatedovernight in media containing wild-type hIL13 exhibited a markedincrease in VCAM-1 (FIG. 4, panels B and H). The pattern of the stainingappeared to be specific for certain areas of the cell surface, comparedto the minimal, homogeneous staining of cells that had not beenincubated with cytokine (FIG. 4, panels A and G). Cells incubated withmutants hIL13.E13I, hIL13.E13K, and hIL13.E13Y, which are unable toinduce TF-1 cell proliferation (FIG. 3), showed less VCAM-1 expressionthan those treated with wild-type hIL13 (FIG. 4, panels C, D, F, and B,respectively). Although mutant hIL13.F113D was not tested, thehIL13.R109D-induced VCAM-1 staining was negligible (not shown),suggesting again the involvement of aa-helix D of the cytokine ineffective signaling through the shared receptor. Cells treated withmutants hIL13.E13R and hIL13.E17K showed an increase in VCAM-1 stainingsimilar to that induced by wild-type hIL13, when compared to theirrespective controls (FIG. 4, panels E and J). Mutant hIL13.E16K appearedto have a superagonistic effect on VCAM-1 expression compared to itswild-type IL13 control (FIG. 4, panels I and H, respectively).

The ability of hIL13 and its mutants to block the cancer-restrictivehIL13 receptor on two different human glioblastoma cell lines wasexamined in cytotoxicity assays using hIL13-PE1E, an extremely potentanti-tumor agent on glioma cells (see Debinski et al. (1996) J. Biol.Chem., 271: 22428-22433). The cytotoxin caused a high level ofcytotoxicty in cultured U-251-MG cells (FIG. 5, panel A) and SNB-19cells (FIG. 5, panel B) when the cells were cultured in the absence of acompeting ligand for the receptor. When cultured in the presence ofhIL13 or any of its A or C helix mutants, the level of cytotoxicity wasreduced even at the highest concentration of cytotoxin used (FIG. 5,panels A and B). IC₅₀s for tests without blocking ligand were 0.1 ng/ml(1.25 pM) for U-251-MG cells and 0.07 ng/ml (0.875 pM) for SNB-19 cells.In contrast, the blocking assay using hIL13 mutants showed their abilityto increase the IC₅₀ by at least 100 times. For concentrations of hIL13or its mutants up to 1000×(by weight) over hIL13-PE1E, no discemabledifferences were detected between these various mutants and wild-typehIL13 in blocking the cytotoxin's activity on the glioma cells (FIG. 5,panels A and B). hIL13.F113D, an aa-helix D mutant, behaved as thewild-type cytokine. In contrast, addition of hIL13.R109D to the cellcultures did not reduce the cytotoxin-induced cytotoxicity. hIL4 did notdisplay any neutralizing activity in these assays.

Other Embodiments

This description has been by way of example of how the compositions andmethods of invention can be made and carried out. Those of ordinaryskill in the art will recognize that various details may be modified inarriving at the other detailed embodiments, and that many of theseembodiments will come within the scope of the invention.

Therefore, to apprise the public of the scope of the invention and theembodiments covered by the invention, the following claims are made.

1. A pharmaceutical composition comprising: a pharmacologicallyacceptable excipient; a purified nucleic acid encoding a polypeptidehaving an amino acid sequence identified by any one of SEQ ID NO's:1-23; and, an effector molecule.
 2. The pharmaceutical composition ofclaim 1, wherein the purified nucleic acid encoding a polypeptide has anamino acid sequence identified by SEQ ID NO:
 1. 3. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid encoding apolypeptide has an amino acid sequence identified by SEQ ID NO:
 2. 4.The pharmaceutical composition of claim 1, wherein the purified nucleicacid encoding a polypeptide has an amino acid sequence identified by SEQID NO:
 3. 5. The pharmaceutical composition of claim 1, wherein thepurified nucleic acid encoding a polypeptide has an amino acid sequenceidentified by SEQ ID NO:
 4. 6. The pharmaceutical composition of claim1, wherein the purified nucleic acid encoding a polypeptide has an aminoacid sequence identified by SEQ ID NO:
 5. 7. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid encoding apolypeptide has an amino acid sequence identified by SEQ ID NO:
 6. 8.The pharmaceutical composition of claim 1, wherein the purified nucleicacid encoding a polypeptide has an amino acid sequence identified by SEQID NO:
 7. 9. The pharmaceutical composition of claim 1, wherein thepurified nucleic acid encoding a polypeptide has an amino acid sequenceidentified by SEQ ID NO:
 8. 10. The pharmaceutical composition of claim1, wherein the purified nucleic acid encoding a polypeptide has an aminoacid sequence identified by SEQ ID NO:
 9. 11. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid encoding apolypeptide has an amino acid sequence identified by SEQ ID NO:
 10. 12.The pharmaceutical composition of claim 1, wherein the purified nucleicacid encoding a polypeptide has an amino acid sequence identified by SEQID NO:
 11. 13. The pharmaceutical composition of claim 1, wherein thepurified nucleic acid encoding a polypeptide has an amino acid sequenceidentified by SEQ ID NO:
 12. 14. The pharmaceutical composition of claim1, wherein the purified nucleic acid encoding a polypeptide has an aminoacid sequence identified by SEQ ID NO:
 13. 15. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid encoding apolypeptide has an amino acid sequence identified by SEQ ID NO:
 14. 16.The pharmaceutical composition of claim 1, wherein the purified nucleicacid encoding a polypeptide has an amino acid sequence identified by SEQID NO:
 15. 17. The pharmaceutical composition of claim 1, wherein thepurified nucleic acid encoding a polypeptide has an amino acid sequenceidentified by SEQ ID NO:
 16. 18. The pharmaceutical composition of claim1, wherein the purified nucleic acid encoding a polypeptide has an aminoacid sequence identified by SEQ ID NO:
 17. 19. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid encoding apolypeptide has an amino acid sequence identified by SEQ ID NO:
 18. 20.The pharmaceutical composition of claim 1, wherein the purified nucleicacid encoding a polypeptide has an amino acid sequence identified by SEQID NO:
 19. 21. The pharmaceutical composition of claim 1, wherein thepurified nucleic acid encoding a polypeptide has an amino acid sequenceidentified by SEQ ID NO:
 20. 22. The pharmaceutical composition of claim1, wherein the purified nucleic acid encoding a polypeptide has an aminoacid sequence identified by SEQ ID NO:
 21. 23. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid encoding apolypeptide has an amino acid sequence identified by SEQ ID NO:
 22. 24.The pharmaceutical composition of claim 1, wherein the purified nucleicacid encoding a polypeptide has an amino acid sequence identified by SEQID NO:
 23. 25. The pharmaceutical composition of claim 1, wherein thepurified nucleic acid molecule encoding a polypeptide has an amino acidsequence identified by any one of SEQ ID NO's: 1 to 23, mutants,peptides, fragments and/or combinations thereof.
 26. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid moleculeencoding at least one polypeptide identified by any one of SEQ ID NO's:1-23 is conjugated to an effector molecule.
 27. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid moleculeencoding about two polypeptides identified by any one of SEQ ID NO's:1-23 are conjugated to an effector molecule.
 28. The pharmaceuticalcomposition of claim 1, wherein the purified nucleic acid moleculeencoding up to about ten polypeptides identified by any one of SEQ IDNO's: 1-23 are conjugated to an effector molecule.
 29. Thepharmaceutical composition of claim 1, wherein the purified nucleic acidmolecule encoding at least one polypeptide identified by any one of SEQID NO's: 1-23 is conjugated to at least one effector molecule.
 30. Thepharmaceutical composition of claim 1, wherein the purified nucleic acidmolecule encoding at least one polypeptide identified by any one of SEQID NO's: 1-23 is conjugated to about two effector molecules.
 31. Thepharmaceutical composition of claim 1, wherein the purified nucleic acidmolecule encoding at least one polypeptide identified by any one of SEQID NO's: 1-23 is conjugated to about five effector molecules.
 32. Thepharmaceutical composition of claim 29, wherein the effector moleculeinhibits growth and/or is cytotoxic for a target cell.
 33. Thepharmaceutical composition of claim 29, wherein the purified nucleicacid molecule encoding a polypeptide identified by any one of SEQ IDNO's: 1-23 binds to an IL-13 and/or IL-13/4 cellular receptor in vivo.33. The pharmaceutical composition of claim 29, wherein the purifiednucleic acid molecule encoding a polypeptide identified by any one ofSEQ ID NO's: 1-23 binds to an IL-13 and/or IL-13/4 cellular receptor invitro.
 34. The pharmaceutical composition of claim 29, whereinadministration of a conjugated molecule comprising a polypeptideidentified by any one of SEQ ID NO's: 1-23 and an effector moleculeinhibit growth of tumor cells.
 35. A method for treating cancercomprising: administering to a patient in need thereof, atherapeutically effective amount of a pharmaceutical compositioncomprising a polypeptide fusion molecule, wherein said moleculecomprises a polypeptide identified by any one of SEQ ID NO's: 1-23conjugated to an effector molecule in a pharmaceutical excipient; and,inhibiting growth of cancer cells.
 36. The method of claim 35, whereinthe pharmaceutical composition inhibits growth of tumor cells expressingIL-13 and/or IL-13/4 receptors in a cancer patient.
 37. The method ofclaim 35, wherein any one of the polypeptides identified by SEQ ID NO's:1-23 inclusive, is covalently linked to an effector molecule.
 38. Themethod of claim 35, wherein the effector molecule is cytolytic for cellsexpressing IL-13 and or IL-13/4 receptor expressing cells.
 39. Themethod of claim 38, wherein the effector molecule is a cytotoxin. 40.The method of claim 38, wherein the cytotoxin is Pseudomonas exotoxinand Diphtheria toxin.
 41. The method of claim 35, wherein a cocktail ofsaid pharmaceutical composition is administered to a patient in need ofsuch therapy.
 42. A kit comprising: a chimeric molecule comprising anIL-13 mutant conjugated to an effector molecule; a pharmaceuticalexcipient; printed instructions.
 42. The kit of claim 42, wherein theIL-13 mutant is identified by any of SEQ ID NO's: 1-23.
 43. The kit ofclaim 42, wherein the effector molecule is selected based on a desiredutility of the kit.
 44. The kit of claim 43, wherein the effectormolecule is a cytotoxin.
 45. The kit of claim 43, wherein the effectormolecule is a detectable label.